Removal of protein A from antibody preparations

ABSTRACT

Antibody preparation purified using immobilized protein A and yet substantially free of protein A that may have solubilized during the purificaiton process. The antibodies include less than 15 ng protein A per mg of antibody, preferably less than 1 ng/mg. Low protein A content is obtained by first contacting the antibodies and solubilized protein A with an ion exchange resin under conditions to adsorb both. The antibodies and protein A are then sequentially eluted under conditions of increasing ionic strength.

This application is a division of application Ser. No. 07/584,677 filedSep. 19, 1990, now abandoned, which was a divisional application ofapplication Ser. No. 07/204,054, filed Jun. 8, 1988 which is now U.S.Pat. No. 4,983,722.

BACKGROUND OF THE INVENTION

1. Field

This disclosure is concerned generally with the purification ofbiologically active proteins and specifically with the reduction ofProtein A contamination in therapeutic antibody preparations.

2. Prior Art

Protein A is a well known substance obtained from Staphylococcus aureus,Cowan Strain I. It has a high degree of antibody specificity and theprotein has long been used to complex with antibodies. Protein A iscommonly used in an immobilized form by being attached to various waterinsoluble support materials. The immobilized protein A is then used tocomplex with soluble antibodies which are subsequently eluted fromantibody-immobilized protein A complexes. Protein A chromatography isthus an excellent method for purifying ascites or tissue culture fluidderived monoclonal antibodies to homogeneity because of its simplicityand high degree of antibody specificity.

If the purified monoclonal antibodies are intended for therapeuticusage, however, a major safety concern is the possible presence ofsolubilized Protein A in the purified therapeutic product. Suchsolubilized Protein A is thought to result from the unintendeddetachment of Protein A from its support material during thepurification process.

Numerous publications link Protein A with toxicity and mitogenicity inanimal models and humans (see, for example, Bensinger et al., J. Biol.Resp. Modif. 3, 347, 1984; Messerschmidt et al., J. Biol. Resp. Modif.3,325, 1984; Terman and Bertram, Eur. J. Cancer Clin. Oncol. 21, 1115;1985; and Ventura et al., Hortobagyl. Cancer Treat Rep. 71,411, 1987).

Unfortunately, to date there have been no assay methods available tomeasure very low amounts of Protein A (i.e., less about than 15 ng ofProtein A per mg of protein) that might be undesireably present in anantibody preparation intended for therapeutic use. Surprisingly, wefound such an assay is now possible. Our assay led to a method ofreducing Protein A contamination in antibody preparations to very lowlevels. The method thus permits the production of therapeutic antibodypreparations using immobilized protein A while assuring a low degree ofProtein A contamination. Details of our assay and purification methodsare described below.

SUMMARY OF THE INVENTION

To evaluate the amount of Protein A column leakage, if any, intomonoclonal antibodies purified with immobized Protein A we 1) developeda Protein A ELISA sensitive to the subnanogram range; 2) used the ELISAto determine Protein A levels in monoclonal antibodies purified byProtein A chromatography; and 3) devised a method to reduce solubilizedProtein A found to be contaminating the antibody preparations. Ourhighly sensitive Protein A ELISA was made possible by using biotinylatedanti-Protein A as the immunoassay label, thus allowing measurement ofless than 15 ng of Protein A per mg of antibody. Our method of reducingthe amount of Protein A in an antibody-Protein A mixture comprisescontacting the mixture with an anion exchange column under conditionssufficient to complex both components of the mixture onto an anionexchange material and then selectively eluting the components bycarefully varying ionic strength under conditions sufficient to assureelution of an antibody preparation substantially free of Protein A (i.e.less than 15 ng of Protein A per mg of antibody). Preferably the ProteinA content is less than about 1 ng per mg of antibody, and thepurification step is accomplished by applying the mixture to an ionexchange column such as a DEAE Trisacryl M or DEAE Sepharose ionexchange material column and then eluting the antibody with a NaClsolution concentration gradient of about 0.025M to 0.25M as describedbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing an ELISA for Protein A at concentrationsranging from 0.02 ng/ml to approximately 10 ng/ml.

FIG. 2 is a graph showing a redrawn portion of the standard curve from0.02 ng/ml to 1.3 ng/ml.

FIG. 3 is a graph showing the column profile for the elution of bothantibody and Protein A under conditions of increasing ionic strength.

SPECIFIC EMBODIMENTS

An enzyme labeled immunosorbent assay (ELISA) for low levels of ProteinA was devised by biotinylating anti-Protein A IgG as follows:

Reagents: FTA Hemagglutination buffer (PBS) was obtained from BBL,Microbiology Systems (Cockeysville, Md.). TMB (tetramethyl benzidine)Microwell Peroxidase Substrate and Peroxidase Substrate Solution B wereobtained from Kirkegaard & Perry Labs, Inc. (Gaithersburg, Md.). Rabbitanti-Protein A IgG (#45F-8806) and bovine albumin were obtained fromSigma (St. Louis, Mo.). HRP-Streptavidin enzyme label was purchased fromZymed Laboratories, Inc. (San Francisco, Calif.) and Biotin-X-NHS biotinlabel compound from Calbiochem (La Jolla, Calif.). Natural Protein AAvidGel F immobilizing protein A was obtained from BioProbeInternational, Inc. (Tustin, Calif.). Recombinant Protein A was obtainedfrom Repligen Corp. (Cambridge, Mass.). DEAE-Trisacryl M ion exchangematerial was obtained from LKB Instruments, Inc. (Gaithersburg, Md.).

Methods: Rabbit anti-Protein A IgG was biotinylated according to thesupplier's instructions. The procedure consisted of addition of 20 ul of20 mg/ml Biotin-X-NHS to a 1 mg/ml antibody solution in 0.1M bicarbonatebuffer followed by gentle agitation for 1 hour at room temperature.Excess Biotin-X-NHS was then removed by dialysis against PBS buffer.

The concentration of the Protein A standard was determined using anextinction coefficient of 1.46 for a 1% solution at 275 nm (see Sjoqistet al., Eur. J. Biochem 29,572, 1972)

Our ELISA (Enzyme labeled immunosorbant assay) was performed as follows:Nunc Immuno Plate (#4-3945, InterLab, Thousand Oaks, Calif.) 96 wellflat bottom ELISA plates were coated with 100 uL of 5 ug/ml anti-ProteinA IgG diluted into 0.05M carbonate, pH 9.6 and incubated overnight at 5°C. The plates were then washed with wash buffer (PBS-0.05% Tween 20),blocked with 200 uL of wash buffer plus 1.5% BSA per well and incubatedfor one hour at 37° C. The plates were washed. Samples and the standarddiluted in the wash buffer plus BSA were added in 100 uL aliquots to theplate and incubated for one hour at 37° C. Wells were set aside thatcontained only 100 uL of diluent to serve as blanks. The samples werediscarded and the plates washed again with wash buffer. To each well 100uL of biotinylated anti-Protein A IgG diluted in wash buffer plus BSA(approximately 1/16,000) was applied and incubated for 1 hr at 37° C.The antibody was discarded and the plates washed. Finally, 100 ul perwell of Streptavidin-HRP was added and the plates were incubated for 1hr at 37° C. The plates were washed and 100 uL of a 1:1 solution of TMBMicrowell Peroxidase Substrate and Peroxidase Substrate Solution B wasadded per well and incubated for approximately 5 minutes at roomtemperature. 100 uL of 1N HCl was added to stop the reaction. Theintensity of yellow color generated was proportional to the amount ofProtein A present and was determined by measuring the absorbance at 450nm on a Dynatech MR600 microtiter plate reader (Dynatech. Burlington,Mass.). The reading for each well was compensated for non-specificcontributions by dividing by the absorbance at 570 nm. The average valveof the blanks was subtracted to eliminate background. Experiments weredone in duplicate on the same ELISA plate and average values utilized inthe data analysis method.

ELISA RESULTS ELISA Sensitivity

Purified Protein A was tested in the ELISA at concentrations rangingfrom 0.02 ng/ml to 10 ng/ml. The results shown in FIG. 1 indicatesaturation at approximately 10 ng/ml. The portion of the standard curvefrom 0.02 ng/ml to 1.3 ng/ml is redrawn in FIG. 2 with expanded scalesand shows that the linear portion of the curve is approximately in therange of 0.05 ng/ml to 0.6 ng/ml.

ELISA Validation

The Protein A ELISA was developed to quantitate Protein A in thepresence of monoclonal antibodies. To determine whether the presence ofa monoclonal antibody in stoichiometric excess would interfere with theassay the following experiment was undertaken. A 1 mg/ml solution of amonoclonal antibody purified to homogeneity by ion exchangechromatography was spiked with 10 ug/ml of purified Protein A. As acontrol and standard the ELISA dilution buffer was also spiked with 10ug/ml Protein A. The mixtures were incubated for 1/2 hour at roomtemperature and then diluted to 20 ng/ml in the assay dilution bufferfor assay. The results are shown in FIG. 2. The two curves are nearlysuperimposable. The results for the standard and spiked monoclonalantibody were 20 ng/ml and 18.3 ng/ml respectively. The difference of 9%can be attributed to experimental error. These results suggest that thepresence of an excess amount of a monoclonal antibody does notsignificantly interfere with the ELISA.

Leakage of Protein A

The Protein A contamination levels of several monoclonal antibodypreparations purified by Protein A chromatography were determined (TableI). Eluate pools 1a to 1d represent back-to-back 0.8 liter Protein Acolumn runs with a 3 column volume purge of 0.2M glycine, pH 2.8 betweenruns. An initial Protein A level of approximately 300 ng/ml dropped toapproximately to 40-100 ng/ml in subsequent runs.

Eluate pools 2 and 3 represent a 1.5 liter Protein A column containing ablend of the immobilized Protein A used to obtain Pool 1 eluates and newimmobilized Protein A. Pool 2 eluate Protein A values are similar toPool 1 but Pool 3 eluate values are considerably less. The amount ofProtein A leakage seems to decrease with column usage.

                  TABLE 1                                                         ______________________________________                                        PROTEIN A LEAKAGE FROM IMMOBILIZED                                            PROTEIN A COLUMNS USED                                                        TO PURIFY A MONOCLONAL ANTIBODY                                                                       Protein A/Monoclonal                                  Eluate Pool #                                                                           Protein A (ng/ml)                                                                           (ng/mg)                                               ______________________________________                                        1a        316.8         630                                                   1b        40.9           61                                                   1c        87.8          114                                                   1d        108.4         131                                                   2a        316.0         238                                                   2b        67.4           46                                                   3a        19.6           28                                                   3b        9.5            11                                                   ______________________________________                                    

EXAMPLES Monoclonal Antibody Purification

As measurable amounts of Protein A were present in monoclonal antibodiespurified by immobilized Protein A, we looked for a method to reduce theProtein A contaminant levels. In an initial experiment, a monoclonalantibody previously purified by Protein A chromatography was spiked withpurified Protein A to a concentration of 0.59 ug Protein A per mgmonoclonal antibody and subjected to DEAE chromatography. The columnprofile is shown in FIG. 3. Protein A eluted at a higher NaClconcentration than the IgG and good separation of the protein elutionpeaks was achieved.

To evaluate further the ability of DEAE chromatography to reduce ProteinA contamination in a monoclonal antibody purified on a Protein A column,a Protein A eluate was chromatographed on DEAE (see Table 2).

                  TABLE 2                                                         ______________________________________                                        DEAE CHROMATOGRAPHY OF A MONOCLONAL                                           ANTIBODY PURIFIED ON IMMOBILIZED PROTEIN A                                                                     Protein A/IgG                                Fraction                                                                              IgG (mg/ml)                                                                              Protein A (ng/ml)                                                                           (ng/mg)                                      ______________________________________                                        Leading 0.69       1.0           1.45                                         edge                                                                          Main    3.13       7.8           2.49                                         peak                                                                          Trailing                                                                              0.45       87.6          194.67                                       edge                                                                          ______________________________________                                    

Significant separation of Protein A from the monoclonal was observed inconfirmation of the spiking experiment. Details of a representativepurification process are shown in the following example.

Monoclonal antibody purification: a IgGl tissue culture fluid derivedanti coagulant Factor VIII monoclonal antibody (designated C7F7) waspurified in the following manner: 1) Tissue culture fluid is clarifiedby filtration, 2) Polyethylene glycol is added (17% w/v) to thesupernatent solution and dissolved. Precipitate is separated and thesolution is discarded. The precipitate is dissolved in 0.05Mtris(hydroxymethyl)aminomethane, 0.15M sodium chloride, pH 8.00, to avolume of 2.5% of the original tissue culture fluid. Dissolvedprecipitate may be stored frozen at -20° C. or colder; 3) Fresh orthawed dissolved precipitate is clarified by centrifugation and/orfiltration; 4) Solution is contacted with Protein A Avidgel F (R) (orequivalent immobilized Protein A) equilibrated with dissolving bufferand washed with same. C7F7 is removed by elution with 0.05M sodiumacetate, 0.15M sodium chloride pH 4.00; 5). The eluate is diafilteredagainst not less than 6 volumes of DEAE equilibration buffer (0.025Msodium chloride, 0.025M tris(hydroxymethyl)aminomethane, pH 8.60); 6)The solution is contacted with DEAE Sepharose (R) or equivalent anionexchange resin (previously equilibrated) and washed with DEAEequilibration buffer. C7F7 is eluted with a sodium chloride gradientfrom 0.025M to 0.25M. Eluate is collected based on A280. The trailingedge is discarded. The trailing edge is defined as less than 20% of themaximum peak A280; 7) Diafilter solution against not less than 6 volumesof phosphate buffered saline (8.9 mM disodium phosphate, 0.7 mMmonosodium phosphate, 1.6 mM monopotassium phosphate, 0.15M sodiumchloride). Using the above methods, the Protein A content of theantibody preparation was reduced to less than 15 ng/mg antibody (i.e.range of Protein A was from 0.9 to 14 ng/mg of antibody.

Protein A levels in several Protein A purified monoclonal antibodypreparations further purified with DEAE chromatography were found to bein the range 0.9 to 14 ng/mg.

DISCUSSION

Numerous ELISAs have been developed for the detection of Protein A inthe nanogram range of sensitivity (Maxim et al., J. Clin. Microbiol.4,418, 1976; Langone et al., J. Immunol. Methods 18,281, 1977; Fey andBurkhard, J. Immunol. Methods 47, 99, 1981; Lofdahl et al., Proc. Nat.Acad. Sci., U.S.A., 80,697, 1983; Olsvik and Berdal, Acta Pathol.Microbiol. Immunol. Scand. Sect. B. Microbiol. 89, 289, 1981; Dertzbaughet al., J. Immunol. Methods 83, 169, 1985; Considine et al., Bios. Rep.6, 933, 1986; Warnes, et al., J. Immunol. Methods, 93, 63, 1986). As ourapplication involves assaying Protein A in the presence of excessamounts of IgG, the assay had to be capable of detecting both freeProtein A and Protein A complexed to the Fc region of IgG. Thus theantibodies used in the ELISA have to be specific for epitopes on theProtein A molecule. The majority of the published Protein A ELISAtechniques are unsuitable for this application because they utilize theFc binding ability of Protein A in the assay (Maxim, et al., 1976;Langone et al., 1977; Fey and Burkhard, 1981; Lofdahl, et al., 1983;Olsvik and Berdal, 1981; Considine, et al., 1986; Warnes, et al., 1986).We have developed an ELISA utilizing an anti-Protein A coating antibodyand biotinylated anti-Protein A as the detection antibody. Thebiotinylated secondary antibody ELISA had an assay sensitivityapproximately one hundred fold greater than a similar assay using analkaline phosphatase labeled reagent (Dertzbaugh et al., 1985). Inaddition, with a sensitivity less than 0.1 ng/ml, the ELISA is five toten fold more sensitive than the most sensitive Protein A system yetpublished (Warnes, et al., 1986). Replacement of the rabbit anti-ProteinA-biotin with rabbit anti-Protein A followed by goat anti-rabbitIgG-biotin might improve the assay sensitivity even more (Warnes, etal., 1986).

Protein A contamination of an affinity column purified monoclonalantibody has been previously reported (Dertzbaugh et al., J. Immunol.Methods 83, 169, 1985) and the Protein A values appear to be similar tothose reported here. The 2-fluoro-1-methyl pyridiniumtoluene-4-sulfonate (FMP) activated gel used to link Protein A in ourstudies is reported to be a more stable linkage than the cyanogenbromide-activated gel (Ngo, Bio/Technology 4, 134, 1986) used byDertzbaugh, et al. The observations that the leakage rates of the twoProtein A matrices are similar and that usage seems to reduce leakage,support the hypothesis that the majority of the Protein A that leachedoff the columns was noncovalently bond to the matrix (Dertzbaugh et al.,J. Immunol. Methods, 1985).

DEAE chromatography was demonstrated to effectively reduce Protein Acontaminant levels in preparations of a mouse monoclonal antibody. Inthe presence of excess rabbit or human IgG, Protein A has been shown toform complexes with the molecular formula of [IgG2ProteinA] or thedimeric structure [(IgG)2ProteinA]2 (Balint et al., Cancer Res. 44, 734,1984; Das et al., Anal. Biochem., 145, 27, 1985). These complexes couldbe separated from IgG by Sepharose CL-6B (Balint et al., Cancer Res. 44,734, 1984) or by gel filtration HPLC. Model complexes formed by addingprotein A to serum or to monomeric IgG have been reported to activateFc-bearing leukocytes and the complement system to generate oxidant andanaphylatoxin activity in vitro (Balint et al., Cancer Res. 44, 734,1984). A purification step, such as DEAE or gel filtrationchromatography, designed to reduce contaminant Protein A levels wouldminimize the possibility of toxic effects in recipients of therapeuticmonoclonal antibodies.

Given the above disclosure and examples, it is thought that numerousvariations will occur to those skilled in the art. Accordingly, it isintended that the above examples should be construed as illustrative andthat the scope of the invention disclosed herein should be limited onlyby the following claims.

We claim:
 1. An antibody preparation prepared from a mixture ofantibodies and protein A, the preparation being made by contacting themixture with an anion exchange material under conditions sufficient toadsorb both the antibodies and the protein A and then sequentiallyeluting the antibodies and protein A under conditions of increasingionic strength, the preparation being substantially free of protein A.2. The preparation of claim 1 wherein the amount of protein A is lessthan about 15 ng per mg of antibody.
 3. The preparation of claim 2wherein the amount of protein A is less than about 1 ng per mg ofantibody.
 4. The preparation of claim 1 wherein the antibody preparationcomprises at least one type of monoclonal antibody.
 5. The preparationof claim 1 wherein the antibody preparation comprises monoclonalantibodies to blood coagulation Factor VIII and the protein A content isless than about 15 ng per mg of the antibody.
 6. The preparation ofclaim 5 wherein the protein A content is less than about 1 ng per mg ofantibody.